Conophylline对ADAM10的调控机制及相关研究

发布时间：2018-06-09 19:59

本文选题：阿尔茨海默症 + ADAM10　；参考：《重庆医科大学》2017年硕士论文

【摘要】：研究背景:ADAM10作为a分泌酶,可使APP代谢为sAPPα,一方面减少毒性Aβ的生成,另一方面其产物sAPPα在脑内具有重要生理功能。本实验前期研究通过中药单体高通量药物扫描发现,促胰岛细胞分化药物Conophylline(CNP)可明显增强ADAM10基因启动子区(-508—-30)的荧光素酶活性。目的:旨在从细胞水平探究Conophylline对ADAM10及AD的作用及调节机制。方法:qPCR、荧光素酶活性实验检测细胞水平CNP对ADAM10转录的影响;Western Blot检测ADAM10、APP代谢蛋白、Tau磷酸化水平;Si-RNA研究CNP对ADAM10的调节元件。结果:(1)Conophylline能有效促进SH-SY5Y、HT22和HEK293细胞株ADAM10 mRNA和蛋白水平的表达。(2)Conophylline可抑制稳定过表达APP细胞株HEK-APP中β分泌酶--BACE1,APP代谢产物β-CTF的表达。(3)Conophylline使LC3(LC3-II)表达增高,提示Conophylline可能通过自噬调节CTFs。(4)Conophylline可抑制内源Total-Tau、p Ser396 Tau、p Ser262 Tau和相关激酶CDK5的表达。(5)Conophylline可激活ADAM10基因核心启动子区不同片段(-508—-300,-467—-300,-444—-300)的荧光活性。其中,对-508—-467区域作用明显。(6)Si-RNA分别抑制ADAM10启动子-508—-300区核心转录因子Sp1、USF1及CAAT-BOX结合元件NF-YB后,发现Conophylline主要通过CAAT-BOX调节元件发挥对ADAM10的调控作用。(7)PI3K抑制剂/自噬抑制剂Wortmannin,能抑制Conophylline对ADAM10的影响。自噬作用可能参与了Conophylline对ADAM10的作用。结论:本研究揭示了一种新的小分子中药单体Conophylline对阿尔茨海默病的保护作用,并对其可能参与的机制进行了探究,为下一步动物水平研究奠定实验基础。
[Abstract]:Background: as a secretory enzyme, 1: ADAM10 can metabolize app to sAPP 伪. On the one hand, it can reduce the formation of toxic A 尾, on the other hand, its product sAPP 伪 has important physiological functions in the brain. In the early stage of this experiment, we found that Conophylline (CNP) can obviously enhance the luciferase activity of ADAM10 gene promoter region (-508-30) by high-throughput drug scanning of traditional Chinese medicine monomer. Aim: to investigate the effects and regulatory mechanisms of Conophylline on Adam10 and AD at cellular level. Methods the effect of CNP on the transcription of ADAM10 was detected by cell level CNP assay. Western blot was used to detect the phosphorylation level of ADAM10APP metabolizing protein, Tau phosphorylation level. The regulatory elements of CNP on ADAM10 were studied by Si-RNA. Results Conophylline could effectively promote the expression of mRNA and protein of ADAM10 in SH-SY5YTH-HT22 and HEK293 cell line. Conophylline inhibited the expression of 尾 -secretory enzyme 尾 -CTF in stable over-expressed app cell line, and the expression of 尾 -CTF, the metabolite of 尾 -CTF, increased in HEK293 cell line. It is suggested that Conophylline may inhibit the fluorescence activity of endogenous Total-Taup Ser396 Taup Ser262 Tau and associated kinase CDK5 by regulating CTFs.4Conophylline by autophagy. Conophylline can activate the fluorescence activity of different fragments in the core promoter region of ADAM10 gene, namely -508-300-467-300-444--300). Among them, after inhibiting the core transcription factor Sp1 USF1 and CAAT-BOX binding element NF-YB, the ADAM10 promoter -508-300 region was significantly inhibited by -508-467 si-RNA. It was found that Conophylline could inhibit the effect of Conophylline on ADAM10 mainly through CAAT-BOX regulatory element. The PI3K inhibitor / autophagy inhibitor Wortmanninin could inhibit the effect of Conophylline on ADAM10. Autophagy may be involved in the effect of Conophylline on ADAM 10. Conclusion: this study revealed the protective effect of a new small molecule Chinese medicine monomer Conophylline on Alzheimer's disease and explored the possible mechanism of its participation in order to lay an experimental foundation for the further study on animal level.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16

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